Cloning and expression of cambialistic Bacteroides fragilis superoxide dismutase gene

TR Number



Journal Title

Journal ISSN

Volume Title


Virginia Tech


A gene coding for the cambialistic superoxide dismutase (SOD) was isolated from a LambdaGEM-11 genomic library of Bacteroides fragilis. In order to generate a complete genomic library, B. fragilis genomic DNA was partially digested with the restriction endonuclease Sau3AI and was ligated to cloning vector, LambdaGEM-11. After in vitro packaging, DNA was used to infect E. coli KW 251. The genomic library was finally established in the plaque population. Recombinant phage DNAs containing the SOD gene were detected by a ³²P-labelled synthetic oligonucleotide with 17 bases. The sequence of this oligonucleotide was deduced from the N-terminal amino acid sequence of B. fragilis FeSOD. Two recombinant phage DNAs were selected based on he results of plaque hybridization. Further analysis with restriction mapping and DNA sequencing revealed that only one recombinant phage DNA contained the SOD gene. Southern hybridization and restriction mapping located the SOD gene in the SalI-BamHI fragment (2.1 kb). Sequence analysis identified the orientation and open reading frame (ORF) of the gene. Translation of ORF revealed that SOD consists of 193 amino acid residues. The size of the deduced polypeptide is consistent with the molecular weight of SOD subunit (MW 21,000). The B. fragilis SOD sequence was compared with those of other SODs. The amino acid residues contributing metal ligands, the hydrophobic shell of the active site, and amino acids at the subunit contact are almost fully conserved in B. fragilis SOD. Expression of SalI-BamHI fragment in E. coli SOD double mutant (sodA, sodB), QC1799, produced an active SOD whose activity zymogram was identical to that of purified B. fragilis SOD. In addition, Western analysis of the expressed protein separated on SDS acrylamide gel also displayed a band identical to the subunit of B. fragilis SOD. However, a larger molecular weight band was also detected. This band migrated closely to the subunit of B. fragilis SOD. This larger peptide may be the product of gene translation from an ATG 21 bases upstream of the ATG start codon of B. fragilis gene. The cambialistic feature of SOD gene product was also confirmed from in vitro and in vivo metal substitution.